Abstract The biochemical basis for the development of motility and fertilizing ability of sperm during their passage through the epididymis is largely unknown. Two new discoveries now should enable us to understand the biochemical basis underlying epididymal sperm maturation. One of the signaling protein identified by gene deletion to play a role in epididymal sperm maturation is the calcium regulated protein phosphatase calcineurin. This knock out study however did not address the mechanisms by which how calcineurin performs this function. Another signaling protein is glycogen synthase kinase 3 (GSK3) which has been the subject of a research focus in my laboratory. We identified GSK3 as an enzyme involved in regulating the sperm specific protein phosphatase PP1?2. Both isoforms of GSK3,? and ?, are present in spermatozoa and they are phosphorylated during epididymal sperm maturation and during motility initiation and stimulation. The only phenotype of mice with global disruption of Gsk3? or selective knock out of Gsk3? in developing spermatocytes and spermatids is male infertility. Mice lacking Gsk3? in testis and sperm are fertile. Caudal epididymal sperm lacking GSK3? resemble immature caput epididymal sperm from normal wild type mice. We have now found that calcineurin and GSK3? are mechanistically interrelated. Surprisingly infertile sperm from calcineurin knock out mice have higher rather than lower ATP levels compared to normal wild type sperm. This finding and studies in somatic tissues suggest that lack of calcineurin contributes to an abnormal increase in mitochondrial potential and ATP production that is not synchronized with the development of motility and fertility potential of sperm in the epididymis. We also found that GSK3? in sperm from calcineurin knock out mice is phosphorylated with low catalytic activity. We have shown that GSK3? is involved in ATP generation through glycolysis. Taken together we suggest that calcineurin and GSK3? were the two missing pieces essential to the understanding the biochemical basis underlying activation of metabolism that accompanies sperm motility and fertility development in the epididymis. This exploratory proposal will examine the mechanistic interrelationship between calcineurin and GSK3 in regulating sperm energy metabolism and how pharmacologic inhibition of these two signaling enzymes in sperm affect fertilization.